Krüppel-like factor 17 upregulates uterine corin expression and promotes spiral artery remodeling in pregnancy Can Wanga, Zhiting Wanga, Meiling Hea, Tiantian Zhoua, Yayan Niua,b, Shengxuan Sunc, Hui Lia, Ce Zhanga, Shengnan Zhanga, Meng Liua, Ying Xud, Ningzheng Donga,b,1, and Qingyu Wua,e,1 aCyrus Tang Hematology Center, Ministry of Education Engineering Center of Hematological Diseases, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, 215123 Suzhou, China; bMinistry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 215006 Suzhou, China; cDepartment of Orthopedics, The Second Affiliated Hospital of Soochow University, 215004 Suzhou, China; dCambridge-Soochow University Genomic Resource Center, Soochow University, 215123 Suzhou, China; and eCardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved June 25, 2020 (received for review February 29, 2020) Spiral artery remodeling is an important physiological process in the In the heart, natriuretic peptide expression is controlled by pregnant uterus which increases blood flow to the fetus. Impaired GATA-4, a zinc finger transcription factor (21, 22). A similar spiral artery remodeling contributes to preeclampsia, a major disease GATA-4–dependent mechanism is critical for cardiac corin ex- in pregnancy. Corin, a transmembrane serine protease, is up- pression. We have shown that human and mouse corin gene pro- regulated in the pregnant uterus to promote spiral artery remodeling. moters contain a conserved sequence that is essential for GATA-4 To date, the mechanism underlying uterine corin up-regulation re- binding and corin expression in cardiomyocytes (23). GATA-4 is mains unknown. Here we show that Krüppel-like factor (KLF) 17 is primarily a cardiac transcription factor but not expressed in the a key transcription factor for uterine corin expression in pregnancy. In uterus (24). It is likely that the transcriptional mechanism control- cultured human uterine endometrial cells, KLF17 binds to the CORIN ling corin expression in the uterus differs from that in the heart. promoter and enhances the promoter activity. Disruption of the To test this hypothesis, we analyzed CORIN promoter activi- KLF17 gene in the endometrial cells abolishes CORIN expression. In ties in endometrial and cardiac cells. We also examined corin mice, Klf17 is up-regulated in the pregnant uterus. Klf17 deficiency and transcription factor expression in decidualized human en- MEDICAL SCIENCES prevents uterine Corin expression in pregnancy. Moreover, Klf17- dometrial cells and mouse pregnant uteruses. We identified deficient mice have poorly remodeled uterine spiral arteries and de- Krüppel-like factor 17 (KLF17) as a key transcription factor for velop gestational hypertension and proteinuria. Together, our results uterine corin expression. In mice, Klf17 deficiency prevented reveal an important function of KLF17 in regulating Corin expression Corin expression in the pregnant uterus. Klf17-deficient mice and uterine physiology in pregnancy. developed pregnancy-induced hypertension and proteinuria. These findings indicate an important role of KLF17 in Corin corin | KLF17 | pregnancy expression and uterine physiology in pregnancy. Results n pregnancy, uterine spiral arteries undergo a remodeling Iprocess during which smooth muscle cells in the vessel wall and Human CORIN Promoter Activity in Uterine and Cardiac Cells. Previ- the lumen-lining endothelial cells are replaced by placenta- ously, we identified a conserved GATA-binding sequence in the CORIN derived trophoblasts (1). The remodeled spiral arteries are human promoter required for corin expression in highly dilated, thereby lowering maternal vascular resistance and increasing blood supply to the fetus. Preeclampsia is a disease Significance afflicting ∼5 to 8% of pregnant women and causing maternal and fetal deaths (2). Impaired spiral artery remodeling is a major In pregnancy, blood vessels in the uterus increase in size, pro- factor in the pathogenesis of preeclampsia (2–4). To date, the viding more blood supply and nutrients to the growing fetus. molecular mechanisms underlying spiral artery remodeling are Corin is an enzyme that boosts this process. In this study, we not fully understood. sought to understand how uterine corin production is controlled. Atrial natriuretic peptide (ANP) is a cardiac hormone that In experiments with human uterine cells and pregnant mice, we identified a protein, called Krüppel-like factor 17 (KLF17), which regulates salt-water balance and blood pressure (5). It is syn- turns on the corin gene in the pregnant uterus. In genetically thesized as a precursor, i.e., pro-ANP, which is converted to modified mice, Klf17 deficiency prevents uterine corin produc- mature ANP by corin, a transmembrane serine protease highly tion, decreases uterine vessel size, and causes high blood pres- expressed in the heart (6, 7). In addition to the heart, corin and sure in pregnancy. These results show that KLF17 is a gene- – ANP are expressed in noncardiac tissues, such as kidneys (8 10), controlling protein important for uterus function in pregnancy. hair follicles (11, 12), and developing bones (13, 14). Recently, we and others have shown that corin is up-regulated in the Author contributions: C.W., Z.W., N.D., and Q.W. designed research; C.W., Z.W., M.H., T.Z., pregnant uterus, where corin and ANP promote trophoblast in- Y.N., S.S., H.L., C.Z., S.Z., and M.L. performed research; Y.X. contributed new reagents/ analytic tools; C.W., Z.W., M.H., T.Z., Y.N., S.S., H.L., M.L., Y.X., N.D., and Q.W. analyzed vasion and spiral artery remodeling (15, 16). In pregnant mice, data; and C.W., N.D., and Q.W. wrote the paper. corin and ANP deficiency impairs spiral artery remodeling, The authors declare no competing interest. causing gestational hypertension and proteinuria, a preeclampsia- This article is a PNAS Direct Submission. like phenotype (15, 17, 18). In humans, reduced corin messenger Published under the PNAS license. RNA (mRNA) and protein levels and deleterious CORIN variants 1To whom correspondence may be addressed. Email: [email protected] or have been reported in preeclamptic women (15, 19, 20). These data [email protected]. indicate that uterine corin up-regulation is a part of the physio- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ logical response in normal pregnancy. It remains unknown, how- doi:10.1073/pnas.2003913117/-/DCSupplemental. ever, how corin expression is regulated in the pregnant uterus. First published July 27, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2003913117 PNAS | August 11, 2020 | vol. 117 | no. 32 | 19425–19434 Downloaded by guest on September 29, 2021 cardiomyocytes (23). In this study, we analyzed a 405-bp CORIN we treated ovariectomized C57BL/6 mice with estrogen or proges- promoter fragment containing the GATA-binding sequence terone (SI Appendix,Fig.S2A). By qRT-PCR, we found increased (Fig. 1A). In a luciferase assay, the promoter activity was de- uterine Corin mRNA levels in progesterone-treated, but not tected in human uterine endometrial AN3-CA cells and murine estrogen-treated, mice compared with that in vehicle-treated mice HL-1 cardiomyocytes, in which endogenous corin expression was (Fig. 2A). We next analyzed the uterine expression of all 17 Klf reported (25, 26) (Fig. 1 B and C). Within this promoter frag- genes by qRT-PCR in ovariectomized mice treated with proges- ment, an E-box–binding site and two KLF-binding sites were terone. We found increased uterine mRNA levels of Corin, Klf2, located up-stream of the GATA-binding site (Fig. 1A and SI Klf9, Klf15,andKlf17, but not the of remaining Klf genes (Fig. 2B Appendix, Fig. S1). Deletion of the E-box–binding site had little and SI Appendix,Fig.S2B). We confirmed these results by qRT- effect on the CORIN promotor activity in AN3-CA and HL-1 PCR analysis of uterus tissues from pregnant C57BL/6 mice at cells (Fig. 1 A–C). Further deletion of the two KLF-binding sites different gestational (G) days (Fig. 2C). These results indicate that abolished the promoter activity in AN3-CA, but not HL-1, cells Klf2, Klf9, Klf15, and Klf17 are possible candidates responsible for (Fig. 1 A–C), suggesting a possible role of the KLF-binding sites corin up-regulation in the pregnant uterus. in corin expression in uterine endometrial cells. To verify our results, we mutated the KLF-binding sites, indi- Effects of KLF Expression on CORIN Promoter Activity in Uterine Cells. vidually or together (Fig. 1D). In the luciferase assay, the pro- To examine the function of the KLF candidates, we cotrans- moter fragment with mutations in individual KLF-binding sites fected AN3-CA cells with CORIN promoter constructs and had reduced activities in AN3-CA cells (Fig. 1E). When both of plasmids expressing human KLF2, KLF9, KLF15, and KLF17 the KLF-binding sites were mutated, the promoter activity was proteins. Increased promoter activities were found when plas- abolished in AN3-CA, but not HL-1, cells (Fig. 1 E and F). mids expressing KLF2 and KLF17, but not KLF9 and KLF15, Conversely, mutations in the GATA-binding site abolished the were cotransfected with a promoter construct with the KLF- promoter activity in HL-1, but not AN3-CA, cells (Fig. 1 G–I). binding sites (Fig. 3 A and B). By Western blotting, we con- These results indicate that corin expression in uterine endometrial firmed recombinant KLF expression in the transfected cells cells is controlled by a transcriptional mechanism involving KLF, (Fig. 3C). In these experiments, the promoter activities were not but not GATA, transcription factor(s). increased if the cotransfection was done using the CORIN pro- moter constructs with the KLF-binding sites deleted (Fig. 3D)or Uterine Corin and KLF Expression in Progesterone-Treated and Pregnant mutated (Fig.